Project Summary/Abstract Tuberculosis is a leading cause of death worldwide, infecting 1/3 of the global population. However, the complex chemical composition of the mycobacterial cell wall, robust resilience of Mycobacterium tuberculosis (Mtb) in harsh host environments, and ever-increasing rates of antibiotic resistance have hindered effective treatment of this disease. Mycobacterial proteases, while understudied, represent a class of potential novel drug targets due to their well-conserved nature and importance to cellular physiology. Although the highly conserved serine protease HtrA is dispensable for growth in most other bacteria, Mtb appears to require HtrA activity in multiple contexts. Broadly, this proposal seeks to define how the various functions of HtrA contribute to 1) Mtb's response to environmental stress and 2) the regulation of the mycobacterial cell cycle. Aim 1 interrogates HtrA's role in the mycobacterial stress response, taking a genetic approach to delineate the functions of each of HtrA's domains. In this aim, I will assess the contributions of HtrA's unique cytoplasmic domain and its serine-based proteolytic activity, using a combination of genetics and biochemistry to identify HtrA's binding partners and substrates. Aim 2 investigates HtrA's non-proteolytic role in cell cycle regulation, using multiple complementary strategies to identify cell wall enzymes with which HtrA interacts and define the cell cycle pathways in which HtrA operates. To accomplish this, I will validate putative HtrA binding partners at the cell wall interface, track HtrA kinetics with time-lapse microscopy, and uncover HtrA's genetic interactions with pathway-specific drugs. Together, these aims define HtrA in two different contexts ? as a protease crucial for combating environmental stress and as a novel cell cycle regulator. This work will provide insight into the unprecedented essentiality and diverse functions of mycobacterial HtrA, as well as the unique mechanisms that govern the growth and survival of Mtb. Ultimately, this work has the potential to fill large gaps in knowledge of Mtb's persistence in the context of human infection and provide novel targets for antimycobacterial drugs.